Bacillus thuringiensis belongs to the large group of gram-positive, aerobic, endospore-forming bacteria. Unlike other very closely related species of Bacillus such as B. cereus or B. anthracis, the majority of the hitherto known Bacillus thuringiensis species produce in the course of their sporulation a parasporal inclusion body which, on account of its crystalline structure, is generally referred to also as a crystalline body. This crystalline body is composed of insecticidally active crystalline protoxin proteins, the so-called .delta.-endotoxins.
These protein crystals are responsible for the toxicity to insects of Bacillus thuringiensis. The .delta.-endotoxin does not exhibit its insecticidal activity until after oral intake of the crystalline body, when the latter is dissolved in the intestinal juice of the target insects. In most cases the actual toxic component is released from the protoxin as a result of proteolytic cleavage caused by the action of proteases from the digestive tract of the insects.
The .delta.-endotoxins of the various Bacillus thuringiensis strains are characterized by high specificity with respect to certain target insects, especially with respect to various Lepidoptera, Coleoptera and Diptera larvae, and by a high degree of activity against these larvae. A further advantage in using .delta.-endotoxins of Bacillus thuringiensis resides in the fact that the toxins are harmless to humans, other mammals, birds and fish.
With the introduction of genetic engineering and the new possibilities resulting from it, the field of Bacillus thuringiensis toxins has received a fresh impetus. For example, it is known that many naturally-occurring strains possess more than one insect toxin protein, which may account for a wide spectrum of insecticidal activity of those strains. However, with the ability to genetically transform Bacillus it is possible to create recombinant strains which may contain a chosen array of insect toxin genes obtained by isolation and cloning from naturally-occurring sources. Such recombinant strains can be made to provide whatever spectrum of insecticidal activity might be desired for a particular application, based upon a knowledge of the insecticidal activity of individual toxin proteins. Furthermore, it is also possible to create recombinant toxin proteins which have a chosen combination of functions designed to enhance the degree of insecticidal activity against a particular insect or insect class, or to expand the spectrum of insects against which the toxin protein is active.
The various naturally-occurring insecticidal crystal proteins from Bacillus thuringiensis have been classified based upon their spectrum of activity and sequence similarity. The classification put forth by Hofte and Whiteley, Microbiol. Rev. 53: 242-255 (1989) placed the then known insecticidal crystal proteins into four major classes. Generally, the major classes are defined by the spectrum of activity, with the CryI proteins active against Lepidoptera, CryII proteins active against both Lepidoptera and Diptera, CryIII proteins active against Coleoptera, and CryIV proteins active against Diptera.
Within each major class, the .delta.-endotoxins are grouped according to sequence similarity. The CryI proteins are typically produced as 130-140 kDa protoxin proteins which are proteolytically cleaved to produce active toxin proteins about 60-70 kDa. The active portion of the .delta.-endotoxin resides in the NH.sub.2 -terminal portion of the full-length molecule. Hofte and Whiteley, supra, classified the then known CryI proteins into six groups, IA(a), IA(b), IA(c), IB, IC, and ID. Since then, proteins classified as CryIE, CryIF, CryIG, and CryIX have also been characterized.
It is well known that many .delta.-endotoxin proteins from Bacillus thuringiensis are actually expressed as protoxins. To be insecticidal, these protoxins must first be ingested by the insect, solubilized in the alkaline environment of the insect gut and are proteolytically converted by proteases into a toxic core fragment (Hofte and Whiteley, Microbiol. Rev. 53: 242-255 (1989)). For .delta.-endotoxin proteins of the CryI class, the toxic core fragment is localized in the N-terminal half of the protoxin, whereas the C-terminal portion of the protoxin is proteolytically cleaved to form an active toxin. Chimeric insecticidal proteins can be constructed having novel sequences not found in nature by combining the toxin portion from one .delta.-endotoxin with the protoxin portion of a different .delta.-endotoxin.